Competence to replicate in the unfertilized egg is conferred by Cdc6 during meiotic maturation

Abstract

Meiotic maturation, the final step of oogenesis, is a crucial stage of development in which an immature oocyte becomes a fertilizable egg. In Xenopus, the ability to replicate DNA is acquired during maturation at breakdown of the nuclear envelope by translation of a DNA synthesis inducer that is not present in the oocyte. Here we identify Cdc6, which is essential for recruiting the minichromosome maintenance (MCM) helicase to the pre-replication complex, as this inducer of DNA synthesis. We show that maternal cdc6 mRNA but not protein is stored in the oocyte. Cdc6 protein is synthesized during maturation, but this process can be blocked by degrading the maternal cdc6 mRNA by oligonucleotide antisense injections or by translation inhibition. Rescue experiments using recombinant Cdc6 protein show that Cdc6 is the only missing replication factor whose translation is necessary and sufficient to confer DNA replication competence to the egg before fertilization. The licence to replicate is given by Cdc6 at the end of meiosis I, but the cytostatic factor (CSF) pathway, which maintains large amounts of active Cdc2/Cyclin B2, prevents the entry into S phase until fertilization.

Cdc6 is required for meiotic spindle assembly in Xenopus oocytes

“…The Cdc6 antisense oligonucleotides used in our study has previously been shown to specifically knockdown Cdc6 mRNA and thus Cdc6 protein in Xenopus oocytes. 6 The specificity of Cdc6 antisense oligonucleotides to inhibit Cdc6 protein expression during maturation was further confirmed by protein gel blot analysis (Fig. S2).…”

Factors engaged in reactivation of DNA replication in the nuclei of growing mouse oocytes introduced into the cytoplasm of parthenogenetic one-cell embryos

“…Accumulation of principal replication licensing factors (the proteins of ORC and MCM complexes) was demonstrated in fullygrown and maturing amphibian (Lemaitre et al, 2002;2004;Whitmire et al, 2002) and human (Eward et al, 2004) oocytes. In these studies particular protein fractions (soluble vs. insoluble proteins) were not analyzed.…”

Accumulation and dynamics of proteins of the MCM family during mouse oogenesis and the first embryonic cell cycle

“…This is probably why the ovarian oocytes lack of certain components of pre-RC complexes such as the Cdc6 protein, which is involved in the loading of the MCM proteins on to chromatin. Cdc6 protein is absent in the nuclei of fully-grown oocytes of Drosophila, Xenopus and mouse and is not synthesized before the initiation of maturation (Lemaître et al, 2002;2004;Whitmire et al, 2002, Anger et al, 2005. In contrast, another component of pre-RC complex, the Mcm2 protein, was shown to be stored in the nuclei of fully-grown mouse oocytes (Lemaître et al, 2004).…”

“…To understand how the competence for the DNA replication is built in the activated oocyte it is necessary to know whether the proteins of pre-RC are available in oocytes. The Mcm2-7 proteins have been recently found in the fully-grown oocytes of Xenopus (Whitmire et al, 2002, Lemaître et al, 2002. The Mcm2 protein was found in the fully-grown oocytes of mouse and Drosophila (Lemaître et al, 2004) and in the primary human oocytes (Eward et al, 2004).…”